The long-term objective of this research work is to gain a better understanding of the mechanism of protein biosynthesis in mammalian cells and the roles of different factors in the translation of genetic messages. This study will include the following: (1) Peptide Chain Initiation Factors - We have isolated several protein factors, EIF-1, EIF-1*, Co-EIF-1, EIF-2(A,B), EIF-3 and DF from rabbit reticulocyte ribosomes and have studied their roles in peptide chain initiation. We propose to further purify these factors using conventional methods and study their precise roles in specific steps in the initiation process. (2) Protein Synthesis Inhibitors - We will study the mechanism of regulation of protein synthesis initiation in reticulocyte lysates by heme-regulated inhibitor, HRI and also double-stranded RNA activated inhibitor, dsI. We will also critically examine if these inhibitors undergo changes during aging and thus regulate protein synthesis in aged animals. We will also examine the roles of other protein synthesis inhibitors; translational control RNA (tc RNA), a protein synthesis interference factor (Fx) and Met-tRNAf-deacylase in the regulation of protein synthesis in mammalian cells. In our study, we will use several specific and sensitive peptide chain initiation assay methods recently developed in our laboratory.